This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Kinase phosphorylation with a chemical tag to determine direct in vivo substrates. Eukaryotic protein kinases play a central role in controlling many cellular functions including cell-cell communication, cell-cycle entry/exit, cell morphology and motility, response to UV and O2 stress and many, many other functions. Often multiple kinases are involved in regulation of individual response pathways, making the assessment of the specific role of each kinase very difficult. This is due mainly to the fact that kinases exhibit overlapping substrate specificities which precludes the unambiguous assignment of the direct phosphorylation reaction catalyzed by each kinase in a given pathway. Recently, a new chemical method has been developed for directly tracing kinase substrates using an engineered kinase which accepts an unnatural phosphodonor with a [g-32P) radiolabel. This chemical approach to tracing pathways has been validated by its use in identification of the direct substrates of c-Src which has been under investigation for over 30 years using numerous genetic and biochemical methods. The same substrate tagging method can also be applied to the deconvolution of normal (non-oncogenic) kinase pathways in cells. The specific questions to be addressed are: 1) What substrates are phosphorylated by PINK1, GSK3beta, KSR1, RAF? 2) What are the functions of these phosphorylation events in their respective pathways?